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The role of VUV radiation in the inactivation of bacteria with an atmospheric pressure plasma jet Simon Schneider, Jan-Wilm Lackmann, Dirk Ellerweg, Benjamin Denis, Franz Narberhaus, Julia E. Bandow, and Jan Benedikt* ––––––––– S. Schneider, D. Ellerweg, Jun.-Prof. Dr. J. Benedikt Coupled Plasma-Solid State Systems, Department for Physics und Astronomy, Ruhr-University Bochum, Universitätsstraße 150, 44780 Bochum, Germany E-mail: jan.benedikt@rub.de J.-W. Lackmann, Prof. Dr. F. Narberhaus, Jun.-Prof. Dr. J. E. Bandow Microbial Biology, Department for Biology und Biotechnology, Ruhr-University Bochum, Universitätsstraße 150, 44780 Bochum, Germany B. Denis Institute for Electrical Engineering and Plasma Technology, Ruhr-University Bochum, Universitätsstraße 150, 44780 Bochum, Germany ––––––––– A modified version of a micro scale atmospheric pressure plasma jet (µ-APPJ) source, so-
called X-Jet, is used to study the role of plasma generated VUV photons in the inactivation of
E. coli bacteria. The plasma is operated in He gas or a He/O2 mixture and the X-Jet
modification of the jet geometry allows effective separation of heavy reactive particles (such
as O atoms or ozone molecules) from the plasma-generated photons. The measurements of the
evolution of zone of inhibitions formed in monolayers of vegetative E. coli bacteria, of VUV
emission intensity and of positive ion spectra show that photochemistry in the gas phase
followed by photochemistry products impacting on bacteria can result in bacterial
inactivation. Interestingly, this process is more effective than direct inactivation by VUV
radiation damage. Mainly protonated water cluster ions are detected by mass spectrometry
indicating that water impurity has to be carefully considered. The measurements indicate that
the combination of the presence of water cluster ions and O2 molecules at the surface leads to
the strongest effect. Additionally, it seem that the interaction of VUV photons with effluent of
He/O2 plasma leads to enhanced formation of O3, which is not the case when only O2
molecules and gas impurities at room temperature interacts with plasma generated VUV
photons.
- 2 -
Introduction
Cold atmospheric pressure plasmas (CAP) are increasingly in the focus of researchers
investigating their possible applications in medicine or the food packaging industry.[1, 2] CAP
jets are able to inactivate bacteria or fungi or damage bio-macromolecules..[3, 4, 5] They offer
therefore an alternative to standard sterilization methods there, where thermo-labile and
vacuum-sensitive objects (plastics or living tissues) have to be treated. In addition, several
studies investigate the influence of CAP treatment on wound healing and cancer cells.[6, 7]
These plasmas produce positive and negative ions, (V)UV radiation, and reactive radical
species, which interact with the treated surface. The effects of different plasma-generated
species on the treated systems are a topic of current scientific discussions.[1] The role of
reactive oxygen species (ROS) has been stressed by several authors as key molecules
affecting vegetative prokaryotic and eukaryotic cells.[8, 9] More recently, the combination of
ions and ROS has also been discussed.[2, 10] Capacitively coupled atmospheric pressure plasma
jet (APPJ) sources operated with He with some addition of O2 (≤ 1%) are known to be
efficient sources of ROS, particularly oxygen atoms, ozone molecules (O3), or singlet delta
oxygen metastables O2(a1∆g). Measurements and modeling have been reported for a coaxial
jet with a 1 cm diameter inner electrode and a 1 mm electrode gap, a parallel plate jet with 1
mm electrode separation and 1 mm electrode width, or for sources with an electrode width
larger than 1 mm.[11, 12, 13] The plasma dynamics and plasma chemistry in these discharges
have also been modeled by several authors.[14, 15] These works show that densities of above
mentioned ROS are around 1015 cm−3 in the effluent of these jets and can be tuned by
adjusting O2 concentration, applied power, gas flow, and jet-substrate distance. This kind of
plasma is a promising tool for treatment of living tissues or for antibacterial treatment of
surfaces at atmospheric pressure. The knowledge of ROS densities and, therefore, also the
fluxes could be used to evaluate quantitatively the effects of ROS for example on vegetative
- 3 -
bacteria. With respect to the surface being treated, these sources are remote sources. The
plasma is confined between electrodes and only a neutral plasma effluent without charged
species reaches the surface. It is probably the absence of the flux of charged species to the
surface that makes the treatment of bacteria or living tissues less effective than in low-
pressure reactors where the substrate is in direct contact with the plasma. However, it allows
the fundamental study of the effects of different plasma components on the living cells. An
unknown factor is the effect of VUV photons, which are possibly produced in the plasma in
addition to reactive particles. These photons can propagate unabsorbed through He
atmosphere, irradiate the treated surface and induce uncontrolled radiation damage. We report
in this article the results of a study on the role of VUV photons in inactivation of vegetative
Escherichia coli bacteria. This study is performed with a modified microplasma jet, which
allows well-defined separation of plasma-generated VUV photons and reactive particles.
Experimental Setup
The µ-APPJ has a very simple geometry. It is formed by two stainless steel electrodes (length
30 mm, thickness 1 mm) with a separation of 1 mm, and two glass plates, which are glued to
the electrodes on the side and confine the inter electrode volume. The well-controlled flow
conditions without admixture of surrounding ambient atmosphere and a very good optical
access to the plasma is maintained in this way. The plasma is generated in He gas flow of up
to 5 standard liters per minute (slm) with small concentrations (<1.5 %) of some reactive gas
(in this case O2). A typical α-mode discharge is formed when sinusoidal driving voltage with
root-mean-square value of 150-270 V (frequency 13.56 MHz) is applied to the electrodes.
This source has been described and studied in the past and quantitative measurements of O
and O3 densities as function of O2 concentration, applied power, and distance to the jet are
available.[11, 16, 17] Additionally, this jet geometry with He/O2 gas mixture has been modeled
and discussed in the literature.[14, 15] The gas temperature in the plasma effluent has also been
- 4 -
measured for a 1% O2 admixture and it was below 34◦C.[11] The temperature is slightly higher
at lower O2 concentrations but drops quickly as the distance from the jet increases.
A modified µ-APPJ, a so-called X-Jet, is used in this work. The nozzle of the µ-APPJ is
extended by two crossed channels as shown in a photograph in Figure 1a. These channels are
formed from glass and metal building blocks with 1 mm thickness, and fixed between two
glass plates together with both electrodes. One channel is a direct extension of the inter-
electrode region (direct channel) and the other channel (side channel) crosses the direct
channel under a 45 degree 3 mm downstream of the end of the electrodes. Both channels have
the same 1x1 mm2 cross section. An additional He flow is applied to the side channel to
divert particles in the plasma effluent from the direct channel into the side channel. This is
possible, because the particle transport is controlled mainly by convection at atmospheric
pressure and the flow velocities used. The diffusion is less important due to high collision
rates. Contrary to particles, VUV and UV photons generated in the plasma can propagate
further through the direct channel (also filled with He), cf. the scheme in Figure 1b. Only a
very small fraction (<1.5%) of photons is reflected or scattered into the side channel.
Therefore, operation of the X-Jet with additional He flow through the side channel leads to
effective separation of the plasma-generated reactive particles (for example O atoms or O3
molecules which are emanating from the side channel) and plasma generated photons, which
propagate through the direct channel. Additionally, the experiments are performed in a
chamber with controlled He atmosphere (volume ~ 8 liters) to minimize the influence of
ambient atmosphere. The substrate was always placed perpendicular to the axis of the
corresponding channel used for treatment at a distance of 4 mm. The details regarding the
separation of reactive particles and photons, the results of the simulation of the
convection/diffusion transport in the X-Jet, and the testing of the X-Jet performance in
etching experiments of plasma polymer films, emission spectroscopy measurements in the
- 5 -
115 - 875 nm wavelength range, as well as treatment of bacteria in their vegetative form can
be found elsewhere.[18]
Combined and separate effects of the reactive heavy particles and the VUV and UV radiation
of the plasma effluent on bacteria or other substrates can now be studied in the following
ways: i) photons and reactive particles are applied together: An X-Jet without additional He
flow in the side channel will result in the transport of both reactive particles and photons
through the direct channel. ii) Reactive particles only: The same He flow is used in both
channels. The additional flow through the side channel will push the heavy particles from the
plasma effluent into the side channel as demonstrated in Figure 1b. The flow rates through
both channels after the crossing are the same due to the symmetry of this geometry. The flux
of ROS at the exit of the side channel is expected to be similar to the ROS flux at the direct
channel in i), although some differences will occur due to a missing photo-dissociation and
excitation of ROS and O2 after the crossing of both channels (see also discussion of E. coli
treatment later) and due to a slightly asymmetric velocity field across the side channel. The
plasma generated VUV and UV photons cannot enter directly into side channel due to
geometry constrains, which was corroborated by measurements presented later in this article.
iii) VUV and UV only: With the additional He flow, only VUV and UV photons but no ROS
exit the direct channel. Higher He flow in the side channel can be used to make sure that no
ROS from the plasma diffuse into the direct channel.
Measurement of emission intensity
A solar blind VUV and UV detector (PMT-142, effective in the 115 - 450 nm wavelength
range with a maximum relative efficiency at around 220 nm) in an evacuated housing with
MgF2 window has been used to measure the wavelength integrated intensity of VUV and UV
emission from the direct channel of the X-Jet.[19] A 1 mm diameter diaphragm was placed on
the MgF2 window and the jet was always at 4 mm distance from the window to maintain the
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same acceptance angle for each measurement. The same setup has been used in previous
measurements to verify that the photon flux (in this wavelength range) through the side
channel is negligible.[18]
Measurement of ion fluxes
Molecular beam mass spectrometry (MS) sampling system is used to measure positive ions in
the gas phase. The three stages differentially pumped MS sampling system described
previously is used for these measurements.[17, 20] This system is equipped with a rotating
chopper to modulate the molecular beam and maintain vacuum in the MS. This chopper was,
however, not used here because a smaller sampling orifice (20 μm diameter) was installed.
The X-Jet is placed into a small chamber with controlled He atmosphere, which is mounted
directly at the mass spectrometer front plate with the sampling orifice. Comparable conditions
as during the treatment of bacteria are arranged in this way.
Preparation of biological probes
Vegetative E. coli cells have been used as model substrate in this study. The following
procedure has been used to prepare the substrate and evaluate the effects of exposure to
plasma effluents. E. coli K12 liquid cultures were incubated for 18 hours over night at 37 °C
in LB medium.[21] The cultures were diluted to an optical density of 0.05 at 580 nm and were
sprayed onto LB agar plates for 1 second. A monolayer surface coverage with ~2.2×103·cm−2
cell density is achieved in this case. The plates were grown for 1 hour at 37°C before plasma
treatment. After plasma treatment, the sample plates were incubated over night for 18 hours at
37°C to allow survivors to grow. Zones of inhibition were observed where treatment was
lethal. It was checked that treating of the agar plates with plasma before the application of
cells had no effect on bacterial growth. Furthermore, no pH change of the medium occurred
during plasma treatment. We report in the following results of treatment of vegetative E. coli
bacteria by different components of plasma effluent. The main focus is on the role of VUV
and UV photons in the inactivation of bacteria.
- 7 -
Experimental Results and discussion
The big advantage of the X-Jet is that it allows us to separate the effects of plasma-generated
VUV and UV photons from the effects induced by reactive particles (O3, O, impurities,...).
The effects of VUV and UV photons only, reactive particles only, and photons and reactive
particles together have already been studied in E. coli and selected results are shown in
Figure 2.[18] The following observations have been made: i) the VUV and UV photons
generated in plasma had only a weak effect on E. coli survival. No inactivation was visible in
samples treated with the direct channel for 1 and 3 min of the treatment. The zone of
inhibition at the jet axis appeared only after 6 min of the treatment with photons. ii) The
combined treatment and reactive particle-only treatment showed typical dose-effect
relationships. Elongated treatment times resulted in larger zones of inhibition and lower
numbers of colony forming units. In both cases, the whole area of the Petri dish (80 mm
diameter) was affected after 3 min of treatment. iii) Inactivation by the combined treatment
was approximately twice as fast as with the reactive particle-only treatment (cf. Figure 2). The
fast inactivation by reactive particle-only and by the combined treatment is due to the fact that
the µ-APPJ is an effective source of atomic oxygen and ozone. For example, the densities
(concentrations) of O and O3 measured by molecular beam mass spectrometry at 4 mm
distance from the jet under conditions used in this work are 7×1014 cm−3 (~28 ppm) and
5×1014 cm−3 (~ 20 ppm), respectively.[17] The concentration of ozone increases with the
distance and reaches ~56 ppm at 50 mm. The concentrations of both O and O3 can be
expected to be high enough to cause the observed effects. Ozone is known for its bactericidal
activity. Just 5 min of treatment with 0.2 ppm of ozone in water is lethal for E. coli, Bacillus
cereus, or Bacillus megaterium.[22] Moreover, experiments in air with ozone have shown that
it also effectively kills bacteria on agar plates. Ozone concentrations below 1 ppm and
- 8 -
treatment times smaller 100 min have been reported to be effective in killing Staphylococcus
albus, Streptococcus salivarius, and Bacillus prodigiosus.[23] Atomic oxygen also has
detrimental effects on bacteria. It can etch biological material or cause oxidative stress inside
the cell. We have shown that the flux of atomic oxygen from the side channel of the X-Jet
etches a BSA protein layer or a model plasma polymer film of hydrogenated amorphous
carbon with a rate of 30 nm/min and that the area on the surface, which is affected by atomic
oxygen, is limited to a diameter of ~10 mm.[18, 24] This limitation is given by the fast loss of O
in the gas phase in the three body reaction:[25]
O + O2 + He → O3 + He (1)
Regarding the fact that in treatments with reactive particles longer than 6 min the whole Petri
dish (80 mm diameter) was affected and taking into account the short lifetime of O atoms, we
conclude that mainly O3 is responsible for the inactivation of bacteria at large distances from
the jet axis. Simultaneous action of O, O3, O2 metastables, some possible impurities (like OH
from water) and, in the case of combined treatment through the direct channel, the VUV and
UV radiation most likely play a role in bacterial inactivation near to the jet axis.[18]
An interesting effect, which is further studied and discussed in this article, is the fact that the
combined treatment shows effects twice as fast as the treatment with reactive particles without
photons. It was expected to see more effective inactivation when reactive particles and
photons were treating the substrate simultaneously. However, the simultaneous treatment at
the surface is limited only to an area of 2 to 3 mm diameter just underneath the nozzle of the
direct channel. The rest of the substrate is shadowed by the channel structure and no
synergistic effects were expected there. However, Figure 2 shows that the zone of inhibition
of the combined treatment had a diameter of 20 mm after 1 min of treatment whereas the
treatment with reactive particles only, resulted in a 5 mm diameter after the same time.
Additionally, the combined treatment at 3 min has an effect comparable to the reactive
- 9 -
particles-only treatment at 6 min. These observations indicate that the plasma effluent is
changed by the presence of VUV photons. Some photochemistry reactions take place in the
gas phase in the second part of the direct channel and on the way to the substrate, which are
missing in the reactive particle-only treatment. Additionally, the fact that this change takes
place at greater distance from the jet axis hints that additional ozone might be formed. The X-
Jet geometry offers a unique opportunity to study these effects in more detail.
Study of the photochemistry in the X-Jet
The interaction of photons with some particles in the gas phase and its effects on bacteria can
be studied by admixture of these particles into the He flow administered through the side
channel. Here, we tested this using O2 as it has the second highest density in the plasma
effluent (0.6% concentration, consumption below 5% at URMS = 230 V) after He and its
photochemistry leading to the formation of ozone in the stratosphere is well known. Figure 3
shows the comparison of zones of inhibition induced by photons only (He gas is supplied
through the side channel) and by photons and O2 photochemistry products (He gas with
admixture of O2 at different concentrations is fed through the side channel). Conditions in the
plasma are the same (He gas flow 1.4 slm, O2 concentration of 0.6 %, URMS = 230 V). The gas
flow in the side channel at s slm is higher than that of the flow exiting the plasma to make
sure that diffusion of heavy reactive species from the plasma into the direct channel after the
channel crossing is negligible. Treatment times of 1 and 4 min have been used. No inhibition
zones are observed after 1 min of the treatment when no O2 is added into the He flow through
the side channel. Zones of inhibition with a 3.8 mm diameter appear after 4 min of treatment.
These results are consistent with observations presented in Figure 2. Adding 0.8, 4.2 or 20%
of O2 into the He gas flow in the side channel always results in the formation of an inhibition
zone with diameters between 3 and 4 mm. No clear dependence on the amount of added O2 is
observed after 1 min of the treatment. At 4 min diameters increase to 5, 6.5, and 8.5 mm in an
O2 concentration-dependent manor. The same trend was also observed in several additional
- 10 -
measurements with different plasma conditions and gas flows. The results shown in Figure 3
provide direct evidence that photochemistry of O2 in the effluent of the μ-APPJ has a
measurable effect on the inactivation of bacteria on the time scales of minutes.
Based on these result the possible photochemical processes can be discussed. The
photodissociation threshold of O2 molecules is at 5.12 eV (wavelength 242.4 nm) and the
photoionization threshold at 12.07 eV (102.7 nm).[26] An absolutely calibrated emission
spectrum of the jet has been measured in the past down to the wavelength of 115 nm, the cut-
off limit of the MgF2 window.[27] Two atomic oxygen emission lines at 115 nm (1D - 1Do) and
130 nm (3P - 3So) dominate the spectrum. Additionally, a weak emission due to parts of the
Schumann-Runge bands of O2 and a weak H line at 120 nm (2P - 2So) have been observed.
The photons with wavelengths of 115 nm and 130 nm can dissociate O2 molecules. The
photoabsorption cross-section (which is mainly due to photodissociation) of O2 has its
maximum at 140 nm (2×10-17 cm2), its value at 130 nm is only ~3×10-19 cm2.[26] The mean free
path of 130 nm photons is, therefore, more than 20 cm in He atmosphere with only 0.6% of
O2. This is corroborated by the measurements of the VUV emission intensity by the solar
blind detector sensitive in the 115 – 450 nm wavelength range. The intensity is measured at
the direct channel of the X-Jet operated with the additional He flow through the side channel.
The change of the emission intensity in the 115 – 450 nm wavelength range as function of the
O2 concentration in the additional He flow (O2 concentration in the plasma was kept constant
at 0.6 %) is shown in Figure 4. Indeed, only ~ 3 % of the light is absorbed on the 7 mm
distance from the channel crossing to the MgF2 window if 0.6% of O2 are added into the
additional He flow. The absorption increases almost linearly with increasing O2 concentration.
Additionally, the radiances of about 10 μWmm-2sr-1 were measured for both atomic oxygen
lines at the distance of 4 mm from the nozzle of the μ-APPJ operated with 0.6 % of O2 in the
gas mixture.[27] The radiance increases at lower O2 concentrations in the plasma. It is around
30 μWmm-2sr-1 for the 115 nm line and around 15 μWmm-2sr-1 for the 130 nm line at an O2
- 11 -
admixture of 0.1 %.[27] 10 μWmm-2sr-1 at 130 nm wavelength is approximately a flux of
4×1013 cm-2s-1 photons. This flux is orders of magnitudes smaller than a simulated flux of
8×1016 cm-2s-1 of O-atoms to the surface. Taking into account that only a small fraction of
these photons is absorbed (see Figure 4) it is highly improbable that they are responsible for
the faster inactivation observed in Figure 2.
Photons below the 115 nm threshold, which could unfortunately not be measured with our
diagnostics due to the cutting wavelength of the MgF2 window, can be responsible for this
effect. The measurements of other authors on another plasma source operated with He gas
show that the He∗2 excimer continuum in the 58-100 nm range or a strong atomic oxygen line
at 98 nm can radiate from the plasma.[28] Moreover, the absorption cross section for
photoionization of O2 is ~2×10-17 cm2 in the 30-100 nm range giving a mean free path of
around 3 mm in the gas mixture of He with 0.6 % of O2.[26] The following experiment has
been performed to get new insights into the photochemistry of O2 in the effluent. The He gas
without addition of O2 was used as a plasma forming gas in the direct channel of the X-Jet.
The excimer continuum is expected to be the most intense in this case, because the quenching
of the He*2 in collisions with O2 molecules is reduced. Moreover, no O3 is produced in the
plasma. The chamber with controlled He atmosphere is, therefore, not filled with O3, which
otherwise emanates from the side channel, and experiments with longer exposure time can be
performed. The He flow with variable concentration of O2 (total flow 2 slm) is used again in
the side channel of the X-Jet. E. coli bacteria are than treated for 1, 4, and 10 minutes in the
same way as in Figure 3. The resulting zones of inhibition are shown in Figure 5. Very weak
effect is seen after 1 min within a 3.2 mm diameter. The effect gets stronger after 4 min, but
the affected area is similar. This observation can be explained by direct inactivation of
bacteria by VUV photons from the plasma. A relatively large area with 7-8 mm diameter is
inactivated after 10 min of treatment, which cannot be explained by the direct effect of
- 12 -
photons only. Some reactive species have to be generated in the gas phase (or eventually on
the irradiated surface) that are transported with the gas flow further away from the axis of the
direct channel. Addition of O2 into the He flow in the side channel has the following effects:
i) faster inactivation in a larger area is visible after 1 min and 4 min of the treatment compared
to using He gas only, where the higher O2 concentration is more effective, and ii) the affected
area is clearly larger (diameter > 5 mm) and inactivation faster when 4.2 % of O2 are added
into the He flow. Surprisingly, a zone of inhibition with a diameter of ~7 mm and very sharp
boundary is formed after 10 min of treatment in all cases.
These results corroborate again that VUV photons generated by plasma interact with
molecules in the gas phase in such a way that resulting products inactivate E. coli bacteria.
These photochemistry is more effective when the plasma is operated only in He gas without
0.6% of O2 (compare 1 min treatment in Figure 3 and 5). O2 molecules are clearly involved in
the inactivation process or in the photochemistry, because the observed effect is larger at
higher O2 concentrations in the side channel. However, the fact that prolonged treatment leads
to inactivation only in a limited area indicates that O atoms (which would finally react with
O2 forming stable ozone) are not a product of this photochemistry. Generation of ozone
during the prolonged treatment would lead to inactivation on much larger area, as observed in
Figure 2 for the combined treatment and treatment with reactive particles only. This is clearly
not the case here. Moreover, the 7 mm diameter zone of inhibition is formed even when no O2
is added into the gas flow. The photoionization of O2 molecules and gas impurities (mainly
water molecules) is a plausible explanation of results in Figure 5. Ions can be produced by
VUV radiation, they have a short life time due to the recombination with electrons (and can
therefore reach only a limited area on the surface, which will be similar for all ions), and the
results from the literature indicates that they can act on the bacteria on the treated surface.[10]
The molecular beam mass spectrometry can be used to detect these ions. Figure 6 shows
mass spectra of positive ions measured under the conditions from Figure 5. The X-Jet was
- 13 -
placed into a small chamber with controlled He atmosphere to simulate the conditions during
the treatment of bacteria. The ion spectra are for all conditions dominated by protonated water
clusters with 4 (mass 73 amu) and 5 (mass 91 amu) water molecules. No or very weak signals
only at the noise level (below 10 count/s) are detected below the mass of 70 amu. It is
apparent that water can be ionized effectively by VUV photons generated by He plasma and
therefore that we have VUV photons with wavelengths shorter than 115 nm. The water is an
impurity in the chamber and also in the gas lines and in the X-Jet (both during the treatment
of bacteria and during the MS measurements). It adsorbs at the surface when the chamber is
open and no gas flow is applied and desorbs after the start of the experiment. The
measurements shown in Figure 6 are done just few minutes after closing the chamber and the
start of the gas flows and plasma (which is the situation during the treatment of bacteria). The
signal intensities of all ions drop approximately 200 times within one hour of continuous gas
flushing and plasma operation. Addition of O2 into the side channel leads to higher relative
intensities of water clusters with 5 water molecules (masses 90 and 91) and of ions detected at
masses of 95-97 and 114-116. The overall signal is weaker for the conditions with 4.2% of O2
in the side channel, but part of this decrease is probably due to decreasing water concentration
in the system over time. A Signal at the mass 32 (O2+ ion) appears after the addition of O2
molecules into the side channel, but it is very weak at the level below 10 counts/s. The direct
photoionization of O2 molecules seems therefore to play only a minor role. The MS
measurements can now be related to the results in Figure 5. The zone of inhibition with 7 mm
diameter after 10 min of the treatment with He gas without addition of O2 is probably a result
of the presence of water, water cluster ions or eventually also water fragments (such as OH).
These ions or water fragments can be transported with the gas flow and therefore reach areas
which are not irradiated by the VUV photons. The MS measurement does not show
significant formation of new positive ions (such as O2+) by addition of O2. More probably, the
O2 molecules reacting with the ions on bacteria can accelerate the effect, therefore explaining
- 14 -
the faster inactivation rate and larger inhibition zone diameters after 1 and 4 min of treatment.
This observation corroborates the results of Dobrynin et al., who concluded that ions in the
presence of oxygen (or reactive oxygen species) have the highest efficiency to inactivate
bacteria.[10] It is also in agreement with their conclusion that the presence of non-liquid water
leads to the fastest inactivation. In our case, water which is ionized by the VUV radiation can
originate directly from the surface of the wet agar medium or vegetative bacteria.
The results presented here are still preliminary. The MS measurements and the treatment of
bacteria should be performed under conditions with better control of the water impurity level
in the He gas and with the more precise investigation of the time variation of the process.
These measurements are out of the scope of this article at the moment. Still, the results
presented here provide enough information to demonstrate that VUV photons, photochemistry
products and its combination with O2 molecules on bacteria can play an important role in the
atmospheric pressure plasma sterilization.
Conclusion
A modified version of a micro scale atmospheric pressure plasma jet (µ-APPJ) source, so-
called X-Jet, has been used to study the role of VUV photons in the inactivation of E. coli
bacteria. The plasma was operated in He gas and He/O2 mixture and the X-Jet modification of
the jet geometry allows effective separation of heavy reactive particles (such as O atoms or
ozone molecules) from the plasma generated radiation. The results clearly show that plasma
generated VUV photons play a role in the inactivation of bacteria. However, the
photochemistry of the gas phase species (or adsorbed particles) followed by the reaction of
photochemistry products with bacteria seems to be the more important effect than direct
inactivation of cells by VUV radiation damage. Our results indicate that photoionization of
water clusters by plasma generated VUV photons and the subsequent interactions of these
ions with bacteria could explain the effects observed. The addition of O2 into the gas
accelerates the inactivation, however, only very limited photoionization of O2 is detected. It
- 15 -
indicates that a parallel interaction of water cluster ions (and related photochemistry products)
and O2 molecules at the surface accelerates inactivation of bacteria. Additionally, the results
also indicate that interaction of VUV photons with the effluent of He/O2 plasma (containing
for example vibrationally excited molecules or O2 metastables) leads probably to enhanced
formation of O3, which is not the case when only O2 molecules and gas impurities at room
temperature interact with plasma generated VUV photons.
Acknowledgements: The authors thank Volker Schultz-von der Gathen for fruitful discussions
about the operation of the µ-APPJ source. This work has been performed with the support of
the research group FOR1123 approved by the German Research Foundation (DFG). This
work has also been supported by the Research Department Plasmas with Complex
Interactions of the Ruhr-Universität Bochum as well as through a stipend to J.-W. L. from the
Ruhr University Research School.
Received: ((will be filled in by the editorial staff)); Revised: ((will be filled in by the editorial
staff)); Published online: ((please add journal code and manuscript number, e.g., DOI:
10.1002/ppap.201100001))
Keywords: atmospheric pressure plasma jet; bacterial inactivation; mass spectrometry;
photochemistry; plasma sterilization
- 16 -
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Figure 1. a) Photograph of the µ-APPJ source with two crossed channels after the nozzle, the
X-Jet modification. Additional He (or He/O2) flow diverts the plasma effluent into the side
channel. VUV and UV photons propagate in line-of-sight with the plasma through the direct
channel. b) Schematic representation of the gas flows and photon flux in the channel
structure. The overlap of the blue line with the pink region is the area, where the
photochemistry takes place.
Figure 2. 40 by 40 mm details of photographs of Petri dishes with zones of inhibition in E.
coli monolayers after 1, 3, and 6 min of treatment with reactive particles only, VUV and UV
photons only, or treatment with both combined. Details marked by *: the entire plate (80mm
diameter) was affected. Adopted from [18].
- 19 -
Figure 3: 15 by 15 mm details of photographs of Petri dishes with zones of inhibition in E.
coli monolayers after 1 and 4 min of treatment. Cells were exposed to the effluent of the
direct channel with different O2 concentrations added to the He flow fed through the side
channel. The concentration of O2 in the He flow through the side channel varied from 0 to
20%. Plasma conditions: URMS = 230 V, He flow 1.4 slm, O2 concentration 0.6%.
Figure 4: The change of the overall emission intensity in the 115 – 450 nm region, measured
by solar blind UV and VUV detector, as function of the O2 concentration in the side channel.
- 20 -
Figure 5: 15 by 15 mm details of photographs of Petri dishes with inhibition zones in an E.
coli monolayers after 1, 4 and 10 min of treatment. Cells were exposed to the plasma effluent
passing through the direct channel allowing for photochemistry events by streaming He with
O2 admixture though the side channel. The concentration of O2 in the He flow through the
side channel (total flow 2 slm) varied from 0 to 4.2%. Plasma conditions: URMS = 200 V, He
flow 1.4 slm, no O2 added.
Figure 6: Mass spectrometry measurement of positive ions at 4 mm distance from the end of the direct channel (cf. Figure 1b)) in the mass range 70 to 120 amu. The same conditions as in Figure 5 with three different concentrations of O2 in the He flow through the side channel are compared here
- 21 -
The table of contents entry: The role of VUV radiation in the inactivation of bacteria with an atmospheric pressure plasma jet S. Schneider, J.-W. Lackmann, D. Ellerweg, B. Denis, F. Narberhaus, J. E. Bandow, J. Benedikt
The role of VUV photons in the atmospheric pressure plasma treatment of bacteria is
investigated by means of a modified version of a micro scale He/O2 atmospheric pressure
plasma jet (µ-APPJ) source, so-called X-Jet. The X-Jet allows effective separation of heavy
reactive particles such as O or O3 from the plasma-generated photons. The results show that
the impact of photochemistry products on bacteria is more effective than direct inactivation of
cells by VUV radiation damage and indicate that the combination of water impurity and
presence of molecular oxygen at the surface are important for the process.
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